Trim7 does not have a role in the restriction of murine norovirus infection in vivo

Abstract

Trim7 is an E3 ubiquitin ligase that was recently identified as a central regulator of host-viral interactions with both pro-viral and anti-viral activity in cell culture. As an inhibitor, Trim7 overexpression ubiquitinates viral proteins by recognizing C-terminal glutamines that are hallmarks of 3C-like protease cleavage events. Here, we sought to determine the physiological impact of Trim7 in resolving murine norovirus (MNV) infection of mice, as MNV is potently inhibited by Trim7 in vitro. Utilizing two independently derived Trim7-deficient mouse lines, we found no changes in the viral burden or tissue distribution of MNV in both an acute and persistent model of infection. Additionally, no changes in cytokine responses were observed after acute MNV infection of Trim7-deficient mice. Furthermore, removal of potentially confounding innate immune responses such as STING and STAT1 did not reveal any role for Trim7 in regulating MNV replication. Taken together, our data fail to find a physiological role for Trim7 in regulating MNV infection outcomes in mice and serve as a caution for defining Trim7 as a broad-acting antiviral.IMPORTANCEIntrinsic antiviral molecules that restrict viral replication are important drivers of viral evolution and viral tropism. Recently, Trim7 was shown to provide cell-intrinsic protection against RNA viruses, including murine norovirus. Biochemically, Trim7 recognizes the cleavage product of viral proteases, suggesting a novel and broad mechanism to restrict viral replication. Here, we tested whether Trim7 had a physiological role in restricting murine norovirus replication in mice. Unexpectedly, we found no impact of viral replication or innate immune responses during murine norovirus infection. Our findings urge caution in defining Trim7 as a broad antiviral factor in the absence of in vivo evidence.

Keywords: Trim7; murine norovirus; norovirus.

Fig 1

Overview of Trim7-deficient lines used in this study. (A) Cartoon of the mouse Trim7 locus. sgRNAs 1 and 2 were used to generate two Trim7-deficient mouse lines. Trim7^+1/+1 has a one-nucleotide insertion in exon 1 and has been previously reported (19). Trim7Δ/Δ has a 12,373-nucleotide deletion between exons 1 and 7. Also marked are the qPCR primers and probe used for validation. qPCR primer 1 spans the exon 1/2 junction. (B) Representative western blot of Trim7 of heart tissue from the indicated mouse lines demonstrating a lack of specific band at the predicted molecular weight of Trim7 isoforms. (C) Trim7 expression from indicated tissues from wild-type BL6 mice as measured by qPCR and normalized by concurrent quantification of actin transcripts. Dotted line represents the limit of detection, and each dot represents an individual mouse. (D) Trim7 expression in liver relative to wild-type mice after normalization of actin values. Each dot represents an individual mouse. (E and F) Trim7^WT/WT and Trim7^Δ/Δ bone marrow-derived macrophages (BMDMs) were infected with MNV^CW3 (E) or MNV^CR6 (F) (MOI 0.05). Viral production was measured using plaque assays at indicated time points. Data are shown as mean ± S.E.M. from three independent experiments.

Fig 2

Trim7 does not impact the replication or spread of acute murine norovirus infection in vivo. C57BL/6-Trim7^+1/+1 mice (A) or C57BL/6-Trim7^Δ/Δ mice (B) and respective littermate controls were inoculated with 5 × 10⁶ PFU of MNV^CW3 and euthanized 7 days post-infection. (C) C57BL/6-Trim7^+1/+1 mice and littermate controls were infected with 5 × 10⁶ PFU of MNV^CW3 and euthanized 2 days post-infection. Tissue titers for MLN, spleen, liver, colon, and ileum were analyzed via qPCR for MNV genome copies and normalized to actin. Data shown as mean ± S.E.M. from at least three independent experiments with 3–12 mice per group. ns, **P < 0.01, ***P < 0.001, ****P < 0.0001, Mann-Whitney’s test.

Fig 3

Trim7 does not impact the replication or spread of persistent enteric MNV. C57BL/6-Trim7^+1/+1 mice and littermate controls were inoculated with 1 × 10⁶ PFU of MNV^CR6, and MNV genome copies were enumerated from fecal samples 3, 7, 14, or 21 days post-infection via qPCR (A). Twenty-one days post-infection, animals were sacrificed, and MNV burden was assessed by measuring the genome copies in the MLN (B), colon (C), ileum (D), spleen (E), and liver (F) via qPCR. All samples were normalized relative to actin. Data are shown as mean ± S.E.M. from three independent experiments with 3–9 mice per group. ns, **P < 0.01, ***P < 0.001, ****P < 0.0001, Mann-Whitney’s test.

Fig 4

Trim7 does not affect innate immune response to MNV^CW3 infection. C57BL/6-Trim7^+1/+1 mice and littermate controls were inoculated with 5 × 10⁶ PFU of MNV^CW3 and euthanized 7 days post-infection, and the indicated tissues were collected. (A) Ifnb1, (B) IL6, (C) Ifit1, and (D) Cxcl10 transcript copies were determined via qPCR and normalized to actin levels. The data are plotted relative to the average of the quantities in WT mice post-MNV infection. Data are shown as mean ± S.E.M. from three independent experiments with 4–9 mice per group. ns, **P < 0.01, ***P < 0.001, ****P < 0.0001, one-way analysis of variance with Tukey’s multiple comparison test.

Fig 5

STING and STAT1-dependent innate pathways do not mask the role of Trim7 in restricting MNV^CW3 infection. (A–E) C57BL/6-Sting^Gt/Gt Trim7^+1/+1 or littermate C57BL/6-Sting^Gt/Gt Trim7^WT/+1 were inoculated with 5 × 10⁶ PFU of MNV^CW3 and 7 days post-infection, animals were sacrificed, and MNV burden was assessed by measuring the genome copies in the MLN (A), spleen (B), liver (C), colon (D), and ileum (E) via qPCR. MNV genome levels were determined by qPCR and normalized relative to actin transcripts. Data are shown as mean ± S.E.M. from three independent experiments with 11–12 mice per group. ns, **P < 0.01, ***P < 0.001, ****P < 0.0001, Mann-Whitney’s test. (F) STAT1^−/−/Trim7^+1/+1 and littermate control mice were inoculated with 1 × 10³ PFU of MNV^CW3 and monitored daily for survival for 21 days post-infection (12–13 mice per group). Data from five independent experiments, analyzed using the log-rank Mantel-Cox test.